The Yeast Frataxin Homologue (Yfh1) RESULTS
RESULTS
The colonies appeared depigmented and measurement of total cellular haeme revealed global haeme deficiency in the mutant cells (0.01 nmol haeme/mg dry weight) compared with the wild-type (0.2 nmol haeme/mg dry weight). Low-temperature spectra of yfh1 mutant whole cells revealed a virtual absence of b, c and (a+a3) cytochrome signals (Fig. 1). In contrast, a signal from zinc protoporphyrin was observed in the mutant cells, and this signal was further enhanced by zinc supplementation of the growth media. This observation is significant because biosynthesis of Fe-PPIX (haeme) and Zn-PPIX both require porphyrin precursor and ferrochelatase activity, differing only in the final metal insertion step. The prevalence of Zn-PPIX synthesis rather than haeme synthesis in yfh1 cells did not result from increased zinc accumulation in yfh1 cells compared with wild-type cells. Actually, the total zinc content of the cells was lower in yfh1 cells than in wild-type cells (402 µg zinc per g yfh1 cell paste and 457 µg zinc per g wild-type cell paste). A detailed study of zinc metabolism in yfh1 cells will be published elsewhere.
A general problem that has impeded characterization of Yfh1p function is the variability and instability of the phenotypes of the deletion strains. In part this is due to a tendancy to lose functional mtDNA, thereby becoming rho minus or rho zero. A mutator phenotype characterized by increased frequency of secondary nuclear mutations has also been associated with Yfh1p loss of function (19). Therefore, in order to distinguish primary effects from secondary changes ensuing from lack of YFH1, we created a strain in which the sole copy of YFH1 was placed under the control of a galactose inducible promoter. In this strain, regulated expression of Yfh1 allowed correlation of phenotypes with different Yfh1p expression levels. If this strain was grown in raffinose, a non-inducing carbon source, for 24 h, Yfh1p was undetectable by immunoblotting of isolated mitochondria, and the cells exhibited phenotypes similar to the yfh1 strain. However, under these conditions, rho minus conversion or secondary genetic changes were not observed (not shown). As was observed for the deletion strain, cytochromes were undetectable in the low temperature spectra, whereas Zn-PPIX was clearly discerned as a 580 nm absorbance peak (Fig. 2). Cytochrome c was an abundant haeme protein of the intermembrane space was undetectable by blotting in these cells. When the cells were exposed to galactose, the promoter was rapidly induced and Yfh1p expression reached a maximum within 2 h. Cytochromes in general and cytochrome c in particular were recovered, although the time course of recovery seemed delayed with respect to the recovery of Yfh1p levels. Yfh1p was completely restored at the 2 h time point, whereas cytochromes recovery lagged behind (Fig. 2). The critical Yfh1p function involved in haeme formation presumably occurs during this time interval.
Loss of function of Yfh1 affects the final step of haeme synthesis
The final step in haeme biosynthesis involves iron insertion into PPIX and is catalysed by ferrochelatase, the product of the HEM15 gene. The Hem15 protein, localized to the mitochondrial inner membrane, was markedly decreased in yfh1 mitochondria (Fig. 3A). HEM15 mRNA was also lower in the yfh1 mutant than in wild-type cells (Fig. 3B), as also reported by others (20). Other key enzymes of the haeme pathway were unchanged in yfh1 mutant cells: the amounts of Hem1p ( -aminolevulinate synthase) and Hem13p (coproporphyrinogen oxidase) were the same as in the wild-type (Fig. 3A), and the activity of Hem14p (protoporphyrinogen oxidase) was normal (see below). These data suggested that the defect in haeme synthesis in yfh1 mutant cells might be due to low level of ferrochelatase protein. We therefore sought to correct the Hem15p deficiency by using a multicopy plasmid to increase expression of the gene in the yfh1 strain. Surprisingly, the yfh1 cells transformed with YEp351-HEM15 grew even more slowly than the untransformed cells, forming tiny colonies on agar plates one week after transformation (not shown). The total haeme and cytochrome contents of the transformed mutant cells remained much lower than the wild-type cells (not shown). Thus, the cytochrome defect of yfh1 cells does not result from a lack of Hem15p. Moreover, while cytochromes were almost undetectable in yfh1 cells, a peak of Zn-PPIX was clearly apparent in these cells (see below). Zinc is an alternative substrate of ferrochelatase (21). Thus, although Hem15p was expressed at very low levels in the yfh1 mutant, the protein was still produced and probably functional in the mutant (see below).
An unresolved question was why Hem15 levels were low in the yfh1 strain. HEM15 transcription was found to be regulated in an iron-dependent manner, without dependence on Aft1p or Aft2p, the iron regulatory transcription factors (22,23). As shown in Figure 3B, iron addition to the growth media was correlated with a 2–3-fold increase in transcript abundance whether or not Aft1 or Aft2 was present. In the yfh1 strain the transcript was virtually undetectable. The molecular mediators of this iron dependent regulation remain to be determined. It is generally admitted that Aft-dependent genes are upregulated in yfh1 cells because cytosolic iron is low in these cells, most of the iron being sequestered in the mitochondrial compartment (14). In the case of HEM15, an Aft-independent gene, downregulation of transcription was probably again mediated by alterations in cytoplasmic iron levels, but the regulatory pathway appears to be a novel one.
Iron unavailability for haeme synthesis in yfh1 cells
Studies of in vitro haeme synthesis are often difficult to interpret, since ferrochelatase has a high affinity for ferrous iron, and in vivo iron availability to ferrochelatase probably depends on crucial factors related to iron compartmentalization or availability of an electron donor. Therefore, addition of exogenous ferrous ions and protoporphyrin IX (PPIX) to isolated mitochondria will result in haeme synthesis in vitro, even if no haeme synthesis occurred in vivo.
The level of ferrochelatase protein in yfh1 cells was lower than in wild-type cells, although the residual protein level varied depending on the yeast genetic background from roughly 10% (X498-1A) to 25% (S150-2B yfh1) of normal. This residual ferrochelatase protein was active as shown by the presence of Zn-PPIX in the mutants. Furthermore, the residual ferrochelatase in yfh1 mutants was able to mediate haeme formation (as measured by the pyridine haemochromogen method) when iron as ferrous ascorbate (or ferric citrate+NADH) and PPIX were added to isolated mitochondrial membranes. By contrast, haeme in yfh1 cells (X498-1A or S150-2B) was virtually undetectable, leading us to conclude that an additional defect in iron or porphyrin availability to ferrochelatase must exist in these cells. We then developed an in vitro assay to measure endogenous iron availability to ferrochelatase, using permeabilized whole cells (Fig. 4A) or intact mitochondria (Fig. 4B). We used protoporphyrinogen instead of PPIX as the substrate of reaction, and no exogenous metals, so that only endogenous iron or zinc could be incorporated into PPIX to form haeme or Zn-PPIX. Protoporphyrinogen is the substrate of protoporphyrinogen oxidase, an inter-membrane space enzyme, which converts protoporphyrinogen into PPIX. Protoporphyrinogen and haeme are both non-fluorescent, while PPIX and Zn-PPIX are both highly fluorescent. The use of protoporphyrinogen as substrate allowed monitoring with high sensitivity the rate of PPIX and Zn-PPIX synthesis. In the presence of a metal chelator such as EDTA or 8-hydroxyquinoline, haeme and Zn-PPIX formation from PPIX was inhibited, and we measured the rate of PPIX formation from protoporphyrinogen, i.e. protoporphyrinogen oxidase activity. This activity was comparable in both wild-type and yfh1 mutant cells (Fig. 4A, left panel), showing that the porphyrin substrate for ferrochelatase was not the limiting factor for haeme synthesis. When no chelator was added, part of the PPIX synthesized could be used by ferrochelatase to form haeme or Zn-PPIX with endogenous metals. The rate of haeme synthesis can then be calculated as: total PPIX (measured in the presence of a chelator) minus (Zn-PPIX+PPIX) (measured without chelator). In the absence of a chelator, much more PPIX accumulated in yfh1 cells than in the wild-type (Fig. 4A, middle panel), suggesting that PPIX was being produced but not utilized for haeme synthesis in the yfh1 cells. Finally, Zn-PPIX was formed more rapidly in yfh1 cells than in wild-type cells, consistent with the unavailability of iron as a substrate for ferrochelatase (Fig. 4A, right panel). Similar experiments were done with intact isolated mitochondria from the GAL-Yfh1 strain (galactose-dependent expression of YFH1) repressed for YFH1 expression, and induced for 2 h and 5 h with galactose (Fig. 4B). Similar activity of protoporphyrinogen oxidase was observed with all mitochondria samples (Fig. 4B, upper left panel). However, when endogenous metals were not chelated by EDTA, the rate of formation of PPIX from protoporphyrinogen decreased as YFH1 was induced (Fig. 4B, upper right panel), meaning that an increasing part of the PPIX produced was used to form Zn-PPIX and haeme as YFH1 became induced. The rate of Zn-PPIX formation with endogenous zinc was comparable in all the mitochondria samples (Fig. 4B, bottom left panel). In contrast, the availability of endogenous iron to form haeme was very low in mitochondria with repressed Yfh1, and increased after galactose induction, especially after 5 h induction (Fig. 4B, bottom right panel). This result fits well with the data of Figure 2, showing induction of cytochrome synthesis in cells of the same strain after 2 and 5 h induction of YFH1 by galactose. It is interesting to note that induction of Yfh1 expression in Yfh1-deficient cells did not result in immediate restoration of haeme synthesis and cytochrome production: while the level of Yfh1p was comparable to wild-type levels after 2 h induction by galactose, the synthesis of haeme (Fig. 4B) and of cytochromes (Fig. 2) was maximum only 5 h after induction.
Iron precipitation in an inorganic form in yfh1 mitochondria
The observation that iron was unavailable for haeme synthesis in yfh1 cells raised a question of why this should be so. The physical state of the iron was examined in mutant mitochondria using Mössbauer spectroscopy. Figure 5 shows Mössbauer spectra of mitochondria purified from a wild-type (YPH499) recorded at 4.3 K in a small perpendicular field of 20 mT. No Mössbauer signal was visible, indicating an iron concentration therein of lower than 300 µM and a lack of iron accumulation. In contrast, the mitochondria of the isogenic yfh1 mutant (X498-1A) displayed a well-resolved quadrupole doublet at 4.3 K (spectrum not shown) exhibiting an isomer shift =0.53(4) mm s-1, a quadrupole splitting EQ=0.63(1) mm s-1 and a line width =0.57(1) mm s-1 (numbers in brackets correspond to calculated error of last digit). No ferrous iron was observed. These Mössbauer parameters are typical of a high-spin ferric iron bound to oxygen/nitrogen in an octahedral arrangement. Very similar parameters were found for various bacterioferritins at this temperature (24–27). Mitochondria of a yfh1 suppressor strain isolated from X498-1A (sup4+, see Fig. 5) exhibited a doublet with almost the same Mössbauer parameters ( =0.52 mm s-1, EQ=0.67 mm s-1, =0.53 mm s-1; not shown). However, the degree of ferric ion accumulation per gram mitochondria was approximately one-quarter of that found in the original yfh1 strain.
A -value of 0.57 mm s-1 indicates a line-width broadening which can be associated with relaxation or superparamagnetic phenomena. Indeed, further broadening of the Mössbauer lines [ =0.53(4) mm s-1, EQ=0.64(1) mm s-1, =0.73(1) mm s-1] occurred at 1.9 K (Fig. 5). Moreover, the formation of a second unstructured component (42% of absorption area) was observed. In contrast to what was found in bacterioferritins, no indication for a distinct magnetic hyperfine field or a narrow ranged field distribution was visible. This and the features of a high field spectrum (7 T, not shown) are consistent neither with a superparamagnetic transition as observed in bacterioferritins, nor with a magnetic transition of antiferromagnetically µ-oxo-coupled systems. The featureless broadening is best explained by a broad distribution of individual hyperfine fields originating from many magnetically non-equivalent ferric ions. Thus, our data are consistent with the presence of small and very amorphous nano-particles of iron in yfh1 mitochondria. Various attempts to visualize these particles on PAGE failed. The material remained in the wells of the gel, as seen by Fridovich staining (not shown). There were only very small amounts, if any, of protein associated with these particles (0.1 µg protein/µg iron), which could represent non-specific adsorption. Phosphate and iron determination resulted in a Fe/P ratio of 1/2.9 (8). We conclude that iron was essentially present in yfh1 mitochondria as nano-particles of ferric phosphate. In fact, an EXAFS analysis (not shown) supported the structural model of ferric phosphate as the main iron compound of yfh1 mitochondria. A complete analysis of EPR-, Mössbauer spectroscopic and EXAFS data from whole cells and from mitochondria will be published elsewhere.
Suppressor mutations frequently mask the cytochrome defect of yfh1 cells
Some authors described normal cytochrome production in yfh1 cells (28). According to this observation, the cytochrome and respiratory defects reported by others for yfh1 mutants (3) could depend on a particular genetic background, on the growth conditions, or could result from rho minus conversion of the cells. Our results, however, do not support this hypothesis. We constructed a yfh1 shuffle strain where the yfh1 deletion was covered by a shuffle plasmid bearing a wild-type copy of YFH1. Cells of this strain formed isolated, depigmented colonies when plated on YPD+cycloheximide, but did not grow on YPG+cycloheximide plates (not shown). When yfh1 cells from a YPD plate were inoculated in liquid medium with glycerol as the carbon source, growth was delayed by a lag of 1–3 days (not shown). The cells harvested after 5 days on glycerol medium showed a normal cytochrome spectrum, unlike cells grown on raffinose as the carbon source, which were completely depigmented (Fig. 6). Cells from the glycerol culture did not recover the original phenotype of yfh1 (lack of cytochrome) when re-inoculated on a raffinose-based medium (Fig. 6). This result indicates that some inheritable change(s) occurred to the cells during their growth on glycerol. Indeed, yfh1 cells accumulated suppressor mutations with high frequency, which was easily observed on agar plate. When a mat of yfh1 cells was plated onto a YPG plate, numerous colonies grew on a background of non-growing (or poorly growing) cells (Fig. 7). The same observation was made with yfh1 cells from different genetic backgrounds, including YPH499, S150-2B, CM3260 and W303 (not shown). We analysed several suppressor colonies of yfh1 from various genetic backgrounds. Most of the time, the suppressor strains exhibited the same phenotype as presented in Fig. 8. Cells recovered a normal cytochrome content (Fig. 8A) and normal activity of haeme-containing enzymes such as catalase (not shown). The activity of enzymes containing an iron–sulphur cluster remained low (Fig. 8B). Ferrochelatase and cytochrome c levels were increased compared to the original yfh1 mutant (Fig. 8C). Respiratory activity was similar to that of the wild-type (Fig. 8D). Cell iron accumulation decreased compared with the original yfh1 mutant but was still higher than in the wild-type (Fig. 8E), and iron still accumulated in the mitochondria, although to a lesser extent than in the original yfh1 mutant (not shown). Resistance of the cells to oxidative stress was increased (Fig. 8F). The suppressor phenotype of glycerol-growing cells resulted from nuclear mutation(s).
We crossed yfh1 cells showing the suppressor phenotype with an original yfh1 mutant of the same background with the opposite mating type. Features of the diploid were intermediate between the suppressor strain and the non-suppressed deletion strain, indicating semi-dominance of the suppressor mutation (Fig. 8C). Following sporulation and tetrad dissection, the suppressor phenotype was recovered in the tetrads, showing a 2:2 segregation of the suppressor characteristics (Fig. 8C). Such nuclear (semi-dominant) suppressor mutations occurred with high frequency in yfh1 cells submitted to the selection pressure of a non-fermentable carbon source or oxidative stress. The high rate of new suppressor mutations has prevented us from identifying the suppressor gene by complementation. A genomic library was constructed from a yfh1 suppressor strain and used to transform an original yfh1 strain. Transformants were selected on a copper-rich medium allowing growth of suppressors but not of original yfh1 cells. All the colonies analysed were new suppressor strains (not shown).
Thus, the presence of normal cytochrome concentration in some yfh1 strains (28) may result from suppressor mutations rather than from adaptation of the cells to particular growth conditions. Conversely, the lack of cytochromes in yfh1 was not a consequence of rho minus conversion of the cells, since induction of Yfh1 by galactose in the GAL-Yfh1 strain rapidly induced synthesis of all the cytochromes, with concomitant disappearance of Zn-PPIX, as shown above (Fig. 2). We conclude that Yfh1 is required for normal cytochrome synthesis in yfh1 cells, independent from the background and from the tendency of cells to loose mitDNA.
RESULTS
The colonies appeared depigmented and measurement of total cellular haeme revealed global haeme deficiency in the mutant cells (0.01 nmol haeme/mg dry weight) compared with the wild-type (0.2 nmol haeme/mg dry weight). Low-temperature spectra of yfh1 mutant whole cells revealed a virtual absence of b, c and (a+a3) cytochrome signals (Fig. 1). In contrast, a signal from zinc protoporphyrin was observed in the mutant cells, and this signal was further enhanced by zinc supplementation of the growth media. This observation is significant because biosynthesis of Fe-PPIX (haeme) and Zn-PPIX both require porphyrin precursor and ferrochelatase activity, differing only in the final metal insertion step. The prevalence of Zn-PPIX synthesis rather than haeme synthesis in yfh1 cells did not result from increased zinc accumulation in yfh1 cells compared with wild-type cells. Actually, the total zinc content of the cells was lower in yfh1 cells than in wild-type cells (402 µg zinc per g yfh1 cell paste and 457 µg zinc per g wild-type cell paste). A detailed study of zinc metabolism in yfh1 cells will be published elsewhere.
A general problem that has impeded characterization of Yfh1p function is the variability and instability of the phenotypes of the deletion strains. In part this is due to a tendancy to lose functional mtDNA, thereby becoming rho minus or rho zero. A mutator phenotype characterized by increased frequency of secondary nuclear mutations has also been associated with Yfh1p loss of function (19). Therefore, in order to distinguish primary effects from secondary changes ensuing from lack of YFH1, we created a strain in which the sole copy of YFH1 was placed under the control of a galactose inducible promoter. In this strain, regulated expression of Yfh1 allowed correlation of phenotypes with different Yfh1p expression levels. If this strain was grown in raffinose, a non-inducing carbon source, for 24 h, Yfh1p was undetectable by immunoblotting of isolated mitochondria, and the cells exhibited phenotypes similar to the yfh1 strain. However, under these conditions, rho minus conversion or secondary genetic changes were not observed (not shown). As was observed for the deletion strain, cytochromes were undetectable in the low temperature spectra, whereas Zn-PPIX was clearly discerned as a 580 nm absorbance peak (Fig. 2). Cytochrome c was an abundant haeme protein of the intermembrane space was undetectable by blotting in these cells. When the cells were exposed to galactose, the promoter was rapidly induced and Yfh1p expression reached a maximum within 2 h. Cytochromes in general and cytochrome c in particular were recovered, although the time course of recovery seemed delayed with respect to the recovery of Yfh1p levels. Yfh1p was completely restored at the 2 h time point, whereas cytochromes recovery lagged behind (Fig. 2). The critical Yfh1p function involved in haeme formation presumably occurs during this time interval.
Loss of function of Yfh1 affects the final step of haeme synthesis
The final step in haeme biosynthesis involves iron insertion into PPIX and is catalysed by ferrochelatase, the product of the HEM15 gene. The Hem15 protein, localized to the mitochondrial inner membrane, was markedly decreased in yfh1 mitochondria (Fig. 3A). HEM15 mRNA was also lower in the yfh1 mutant than in wild-type cells (Fig. 3B), as also reported by others (20). Other key enzymes of the haeme pathway were unchanged in yfh1 mutant cells: the amounts of Hem1p ( -aminolevulinate synthase) and Hem13p (coproporphyrinogen oxidase) were the same as in the wild-type (Fig. 3A), and the activity of Hem14p (protoporphyrinogen oxidase) was normal (see below). These data suggested that the defect in haeme synthesis in yfh1 mutant cells might be due to low level of ferrochelatase protein. We therefore sought to correct the Hem15p deficiency by using a multicopy plasmid to increase expression of the gene in the yfh1 strain. Surprisingly, the yfh1 cells transformed with YEp351-HEM15 grew even more slowly than the untransformed cells, forming tiny colonies on agar plates one week after transformation (not shown). The total haeme and cytochrome contents of the transformed mutant cells remained much lower than the wild-type cells (not shown). Thus, the cytochrome defect of yfh1 cells does not result from a lack of Hem15p. Moreover, while cytochromes were almost undetectable in yfh1 cells, a peak of Zn-PPIX was clearly apparent in these cells (see below). Zinc is an alternative substrate of ferrochelatase (21). Thus, although Hem15p was expressed at very low levels in the yfh1 mutant, the protein was still produced and probably functional in the mutant (see below).
An unresolved question was why Hem15 levels were low in the yfh1 strain. HEM15 transcription was found to be regulated in an iron-dependent manner, without dependence on Aft1p or Aft2p, the iron regulatory transcription factors (22,23). As shown in Figure 3B, iron addition to the growth media was correlated with a 2–3-fold increase in transcript abundance whether or not Aft1 or Aft2 was present. In the yfh1 strain the transcript was virtually undetectable. The molecular mediators of this iron dependent regulation remain to be determined. It is generally admitted that Aft-dependent genes are upregulated in yfh1 cells because cytosolic iron is low in these cells, most of the iron being sequestered in the mitochondrial compartment (14). In the case of HEM15, an Aft-independent gene, downregulation of transcription was probably again mediated by alterations in cytoplasmic iron levels, but the regulatory pathway appears to be a novel one.
Iron unavailability for haeme synthesis in yfh1 cells
Studies of in vitro haeme synthesis are often difficult to interpret, since ferrochelatase has a high affinity for ferrous iron, and in vivo iron availability to ferrochelatase probably depends on crucial factors related to iron compartmentalization or availability of an electron donor. Therefore, addition of exogenous ferrous ions and protoporphyrin IX (PPIX) to isolated mitochondria will result in haeme synthesis in vitro, even if no haeme synthesis occurred in vivo.
The level of ferrochelatase protein in yfh1 cells was lower than in wild-type cells, although the residual protein level varied depending on the yeast genetic background from roughly 10% (X498-1A) to 25% (S150-2B yfh1) of normal. This residual ferrochelatase protein was active as shown by the presence of Zn-PPIX in the mutants. Furthermore, the residual ferrochelatase in yfh1 mutants was able to mediate haeme formation (as measured by the pyridine haemochromogen method) when iron as ferrous ascorbate (or ferric citrate+NADH) and PPIX were added to isolated mitochondrial membranes. By contrast, haeme in yfh1 cells (X498-1A or S150-2B) was virtually undetectable, leading us to conclude that an additional defect in iron or porphyrin availability to ferrochelatase must exist in these cells. We then developed an in vitro assay to measure endogenous iron availability to ferrochelatase, using permeabilized whole cells (Fig. 4A) or intact mitochondria (Fig. 4B). We used protoporphyrinogen instead of PPIX as the substrate of reaction, and no exogenous metals, so that only endogenous iron or zinc could be incorporated into PPIX to form haeme or Zn-PPIX. Protoporphyrinogen is the substrate of protoporphyrinogen oxidase, an inter-membrane space enzyme, which converts protoporphyrinogen into PPIX. Protoporphyrinogen and haeme are both non-fluorescent, while PPIX and Zn-PPIX are both highly fluorescent. The use of protoporphyrinogen as substrate allowed monitoring with high sensitivity the rate of PPIX and Zn-PPIX synthesis. In the presence of a metal chelator such as EDTA or 8-hydroxyquinoline, haeme and Zn-PPIX formation from PPIX was inhibited, and we measured the rate of PPIX formation from protoporphyrinogen, i.e. protoporphyrinogen oxidase activity. This activity was comparable in both wild-type and yfh1 mutant cells (Fig. 4A, left panel), showing that the porphyrin substrate for ferrochelatase was not the limiting factor for haeme synthesis. When no chelator was added, part of the PPIX synthesized could be used by ferrochelatase to form haeme or Zn-PPIX with endogenous metals. The rate of haeme synthesis can then be calculated as: total PPIX (measured in the presence of a chelator) minus (Zn-PPIX+PPIX) (measured without chelator). In the absence of a chelator, much more PPIX accumulated in yfh1 cells than in the wild-type (Fig. 4A, middle panel), suggesting that PPIX was being produced but not utilized for haeme synthesis in the yfh1 cells. Finally, Zn-PPIX was formed more rapidly in yfh1 cells than in wild-type cells, consistent with the unavailability of iron as a substrate for ferrochelatase (Fig. 4A, right panel). Similar experiments were done with intact isolated mitochondria from the GAL-Yfh1 strain (galactose-dependent expression of YFH1) repressed for YFH1 expression, and induced for 2 h and 5 h with galactose (Fig. 4B). Similar activity of protoporphyrinogen oxidase was observed with all mitochondria samples (Fig. 4B, upper left panel). However, when endogenous metals were not chelated by EDTA, the rate of formation of PPIX from protoporphyrinogen decreased as YFH1 was induced (Fig. 4B, upper right panel), meaning that an increasing part of the PPIX produced was used to form Zn-PPIX and haeme as YFH1 became induced. The rate of Zn-PPIX formation with endogenous zinc was comparable in all the mitochondria samples (Fig. 4B, bottom left panel). In contrast, the availability of endogenous iron to form haeme was very low in mitochondria with repressed Yfh1, and increased after galactose induction, especially after 5 h induction (Fig. 4B, bottom right panel). This result fits well with the data of Figure 2, showing induction of cytochrome synthesis in cells of the same strain after 2 and 5 h induction of YFH1 by galactose. It is interesting to note that induction of Yfh1 expression in Yfh1-deficient cells did not result in immediate restoration of haeme synthesis and cytochrome production: while the level of Yfh1p was comparable to wild-type levels after 2 h induction by galactose, the synthesis of haeme (Fig. 4B) and of cytochromes (Fig. 2) was maximum only 5 h after induction.
Iron precipitation in an inorganic form in yfh1 mitochondria
The observation that iron was unavailable for haeme synthesis in yfh1 cells raised a question of why this should be so. The physical state of the iron was examined in mutant mitochondria using Mössbauer spectroscopy. Figure 5 shows Mössbauer spectra of mitochondria purified from a wild-type (YPH499) recorded at 4.3 K in a small perpendicular field of 20 mT. No Mössbauer signal was visible, indicating an iron concentration therein of lower than 300 µM and a lack of iron accumulation. In contrast, the mitochondria of the isogenic yfh1 mutant (X498-1A) displayed a well-resolved quadrupole doublet at 4.3 K (spectrum not shown) exhibiting an isomer shift =0.53(4) mm s-1, a quadrupole splitting EQ=0.63(1) mm s-1 and a line width =0.57(1) mm s-1 (numbers in brackets correspond to calculated error of last digit). No ferrous iron was observed. These Mössbauer parameters are typical of a high-spin ferric iron bound to oxygen/nitrogen in an octahedral arrangement. Very similar parameters were found for various bacterioferritins at this temperature (24–27). Mitochondria of a yfh1 suppressor strain isolated from X498-1A (sup4+, see Fig. 5) exhibited a doublet with almost the same Mössbauer parameters ( =0.52 mm s-1, EQ=0.67 mm s-1, =0.53 mm s-1; not shown). However, the degree of ferric ion accumulation per gram mitochondria was approximately one-quarter of that found in the original yfh1 strain.
A -value of 0.57 mm s-1 indicates a line-width broadening which can be associated with relaxation or superparamagnetic phenomena. Indeed, further broadening of the Mössbauer lines [ =0.53(4) mm s-1, EQ=0.64(1) mm s-1, =0.73(1) mm s-1] occurred at 1.9 K (Fig. 5). Moreover, the formation of a second unstructured component (42% of absorption area) was observed. In contrast to what was found in bacterioferritins, no indication for a distinct magnetic hyperfine field or a narrow ranged field distribution was visible. This and the features of a high field spectrum (7 T, not shown) are consistent neither with a superparamagnetic transition as observed in bacterioferritins, nor with a magnetic transition of antiferromagnetically µ-oxo-coupled systems. The featureless broadening is best explained by a broad distribution of individual hyperfine fields originating from many magnetically non-equivalent ferric ions. Thus, our data are consistent with the presence of small and very amorphous nano-particles of iron in yfh1 mitochondria. Various attempts to visualize these particles on PAGE failed. The material remained in the wells of the gel, as seen by Fridovich staining (not shown). There were only very small amounts, if any, of protein associated with these particles (0.1 µg protein/µg iron), which could represent non-specific adsorption. Phosphate and iron determination resulted in a Fe/P ratio of 1/2.9 (8). We conclude that iron was essentially present in yfh1 mitochondria as nano-particles of ferric phosphate. In fact, an EXAFS analysis (not shown) supported the structural model of ferric phosphate as the main iron compound of yfh1 mitochondria. A complete analysis of EPR-, Mössbauer spectroscopic and EXAFS data from whole cells and from mitochondria will be published elsewhere.
Suppressor mutations frequently mask the cytochrome defect of yfh1 cells
Some authors described normal cytochrome production in yfh1 cells (28). According to this observation, the cytochrome and respiratory defects reported by others for yfh1 mutants (3) could depend on a particular genetic background, on the growth conditions, or could result from rho minus conversion of the cells. Our results, however, do not support this hypothesis. We constructed a yfh1 shuffle strain where the yfh1 deletion was covered by a shuffle plasmid bearing a wild-type copy of YFH1. Cells of this strain formed isolated, depigmented colonies when plated on YPD+cycloheximide, but did not grow on YPG+cycloheximide plates (not shown). When yfh1 cells from a YPD plate were inoculated in liquid medium with glycerol as the carbon source, growth was delayed by a lag of 1–3 days (not shown). The cells harvested after 5 days on glycerol medium showed a normal cytochrome spectrum, unlike cells grown on raffinose as the carbon source, which were completely depigmented (Fig. 6). Cells from the glycerol culture did not recover the original phenotype of yfh1 (lack of cytochrome) when re-inoculated on a raffinose-based medium (Fig. 6). This result indicates that some inheritable change(s) occurred to the cells during their growth on glycerol. Indeed, yfh1 cells accumulated suppressor mutations with high frequency, which was easily observed on agar plate. When a mat of yfh1 cells was plated onto a YPG plate, numerous colonies grew on a background of non-growing (or poorly growing) cells (Fig. 7). The same observation was made with yfh1 cells from different genetic backgrounds, including YPH499, S150-2B, CM3260 and W303 (not shown). We analysed several suppressor colonies of yfh1 from various genetic backgrounds. Most of the time, the suppressor strains exhibited the same phenotype as presented in Fig. 8. Cells recovered a normal cytochrome content (Fig. 8A) and normal activity of haeme-containing enzymes such as catalase (not shown). The activity of enzymes containing an iron–sulphur cluster remained low (Fig. 8B). Ferrochelatase and cytochrome c levels were increased compared to the original yfh1 mutant (Fig. 8C). Respiratory activity was similar to that of the wild-type (Fig. 8D). Cell iron accumulation decreased compared with the original yfh1 mutant but was still higher than in the wild-type (Fig. 8E), and iron still accumulated in the mitochondria, although to a lesser extent than in the original yfh1 mutant (not shown). Resistance of the cells to oxidative stress was increased (Fig. 8F). The suppressor phenotype of glycerol-growing cells resulted from nuclear mutation(s).
We crossed yfh1 cells showing the suppressor phenotype with an original yfh1 mutant of the same background with the opposite mating type. Features of the diploid were intermediate between the suppressor strain and the non-suppressed deletion strain, indicating semi-dominance of the suppressor mutation (Fig. 8C). Following sporulation and tetrad dissection, the suppressor phenotype was recovered in the tetrads, showing a 2:2 segregation of the suppressor characteristics (Fig. 8C). Such nuclear (semi-dominant) suppressor mutations occurred with high frequency in yfh1 cells submitted to the selection pressure of a non-fermentable carbon source or oxidative stress. The high rate of new suppressor mutations has prevented us from identifying the suppressor gene by complementation. A genomic library was constructed from a yfh1 suppressor strain and used to transform an original yfh1 strain. Transformants were selected on a copper-rich medium allowing growth of suppressors but not of original yfh1 cells. All the colonies analysed were new suppressor strains (not shown).
Thus, the presence of normal cytochrome concentration in some yfh1 strains (28) may result from suppressor mutations rather than from adaptation of the cells to particular growth conditions. Conversely, the lack of cytochromes in yfh1 was not a consequence of rho minus conversion of the cells, since induction of Yfh1 by galactose in the GAL-Yfh1 strain rapidly induced synthesis of all the cytochromes, with concomitant disappearance of Zn-PPIX, as shown above (Fig. 2). We conclude that Yfh1 is required for normal cytochrome synthesis in yfh1 cells, independent from the background and from the tendency of cells to loose mitDNA.
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